NEET MDS Lessons
General Microbiology
BACTERIAL GROWTH
The conversion of a parental cell into two daughters constitutes the bacterial life cycle and the time taken to complete cell cycle is known as generation_time. This is around 15 minutes in vegetative bacteria except mycobacteria.
Bacterial Growth Curve
In the presence of fresh growth medium a bacterium shows following four phases;
The Lag phase -> The Log phase -> The Stationary phase -> The Decline phase
The Lag Phase : short duration , bacteria adapt themselves to new environment
The Log Phase (Exponential Phase) : Regular growth of bacteria occurs The morphology of bacteria is best developed in this phase and organisms manifest typical biochemical characters.
- Most of the cidal Abx work best in this phase
• i.e. Ampicillin
- Best phase for staining bacterial cultures
Chemostat and turbidostat are examples of technique by which this phase can be prolonged.
Stationary Phase : balanced growth and cell division cannot be sustained. The total cell Count remains static till lysis supervenes, but the viable cell count quickly declines.
Decline Phase: death phase. Dyeing bacteria exceed the dividing bacterias.
Enzymes:
Serum lysozyme:
Provides innate & nonspecific immunity
Lysozyme is a hydrolytic enzyme capable of digesting bacterial cell walls containing peptidoglycan
• In the process of cell death, lysosomal NZs fxn mainly to aulolyse necrotic cells (NOT “mediate cell degradation”)
• Attacks bacterial cells by breaking the bond between NAG and NAM.
• Peptidoglycan – the rigid component of cell walls in most bacteria – not found in archaebacteria or eukaryotic cells
• Lysozyme is found in serum, tears, saliva, egg whites & phagocytic cells protecting the host nonspecifically from microorganisms
Superoxide dismutase: catalyzes the destruction of O2 free radicals protecting O2-metabolizing cells against harmful effects
Catalase:
- catalyzes the decomposition of H2O2 into H2O & O2
- Aerobic bacteria and facultative anaerobic w/ catalase are able to resist the effects of H2O2
- Anaerobic bacteria w/o catalase are sensitive to H2O2 (Peroxide), like Strep
- Anaerobic bacteria (obligate anaerobes) lack superoxide dismutase or catalase
- Staph makes catalase, where Strep does not have enough staff to make it
Coagulase
- Converts Fibronogen to fibrin
• Coagulase test is the prime criterion for classifying a bug as Staph aureus – from other Staph species
• Coagulase is important to the pathogenicity of S. aureus because it helps to establish the typical abscess lesion
• Coagulase also coats the surface w/ fibrin upon contact w/ blood, making it harder to phagocytize
Variant Forms of Bacteria
Prortoplast ; surface is completely devoid of cell wall component,
Spheroplast : Some residual cell wall component is present
Autoplast: protoplasts which are produced by the action of organisms’ own autolytic enzymes.
L Form: replicate as pleomorphic filtrable elements with defective or no cell wall These are designated as L forms after the Lister Institute where these were discovered by Klineberger-Nobel.
Bacterial Spores: Gram positive bacilli and actinomycetes form highly resistant and dehydrated forms which are called as endospores. The surrounding mother.cell which give rise to them is known as Sporangium. These endospores are capable of survival under adverse conditions
Structure :smooth walled and ovoid or spherical.
In bacilli, spores usually fit into the normal cell diameter except in Clostridium where these may cause a terminal bulge. (drum stick ) or central. , these look like areas of high refractilitv under light microscope.
Germination : This is the process of converting a spore into the vegetative cell. It occurs in less than 2 hours and has three stages:Activation, Germination, Outgrowth
GENETIC VARIATION
Two methods are known for genetic variation in bacteria: mutation and gene transfer.
Mutation : Any change in the sequence of bases of DNA, irrespective of detectable changes in the cell phenotype. Mutations may be spontaneous or induced by various agents which are known as mutagens.
Spontaneous Mutations: Arise from enzymatic imperfections during DNA replications or with transient insertions of transposable elements.
Induced Mutations: Mutation by physical and chemical mutagens.
Physical mutagens ultraviolet rays and high-energy ionizing radiations. The primary effect of UV rays on DNA is the production of pyrmidine dimers whereas ionizing radiations cause single_stranded breaks the DNA molecules.
Chemical mutagens :Affecting nucleotide sequence
(i) Agents which cause error in base pairing (e.g. nitrous acid and alkylating agents).
(ii) Agents which cause errors in DNA replication (e.g. acridine dyes such as acridine orange and profiavine).
(iii) Base analogs which are incorporated into DNA and cause replication errors (e.g. 5-bromouracil)
Gene Transfer
Transformation: Uptake of naked DNA
Transduction : Infection by a nonlethal bacteriophage
Conjugation : Mating between cells in contact
Protoplast fusion
Transformation: Gene transfer by soluble DNA is called as transformation. it requires that DNA be absorbed by the cell, gain entrance to the cytoplasm and undergo recombination with the host genome.
Artificial Transformation(transfection) :Some of the bacteria (such as Escherichia coli) resist transformation until they are subjected to some special treatment such as CaCl2 to make the bacterium more permeable to DNA. Such modified cells can also take up intact double stranded DNA extracted from viruses or in the shape of plasmids. Though the process is same as transformation, it is 9 as transfection because it results in infection by an abnormal route
Transduction :The type of gene transfer in which the DNA of one bacterial cell is introduced into another bacterial cell by viral infection is known as transduction. This introduces only a small fragment of DNA. Because the DNA is protected from damage by the surrounding phage coat, transduction is an easier to perform and more reproducible process than transduction. ,
Two types of transduction are known.
- Generalized transduction When a bacteriophage picks up fragments of host DNA at random and can transfer any genes
- Specialised transduction: phage DNA that has been integrated into the host chromosome is excised along with a few adjacent genes, which the phage can then transfer.
After entry into the host cell, the phage DNA gets incorporated into the host chromosome in such a way that the two genomes are linearly contiguous (lysogeny). The phage genome in this stage is known as prophage, The host cell acquires a significant new property as a consequence of lysogeny because it becomes immune to infection by homologous phage. This is hence called as lysogenic conversion and endow toxigenicity to Corynebacterium diphtheriae
Abortive Transduction :phage DNA fails to integrated into the host chromosome, the process is called as abortive transduction The phage DNA does not replicate and along with binary fission Of the host it goes into one of the daughter cells.
Conjugation :This is defined as the transfer of DNA directly from on bacterial. .cell to another by a mechanism that requires cell-to-cell contact.
The capacity to donate DNA depends upon the possession of the fertility (F) factor. The F pili also retard male-male union. Concomitant with effective male-female pair formation, the circular DNA bearing the F factor is converted to a linear form that is transferred to the female cell in a sequential manner. DNA replication occurs in the male cell and the newly synthesized, semiconserved DNA molecule remains in the male. This ensures postmating characters of the male.
Conjugation in Different Bacteria: Unusual form of plasmid transfer, called phase mediated conjugation has been reported to occur with some strains of Staphylococcus aureus.
Protoplast Fusion: Also called as genetic transfusion. Under osmotically buffered Conditions protoplast fusion takes place by joining of cell membrane and generation of cytoplasmic bridges through which genetic material can be exchanged.
Transposons: Transposons Tn are DNA sequences which are incapable of autonomous existence and which transpose blocks of genetic material back and forth between cell Chromosome and smaller replicons such as plasmids. insertion sequences (IS ) are another similar group of nucleotides which can move from one chromosome to another
Genetic material. IS and Tn are collectively also known as transposable elements or Jumping genes. These are now recognised to play an important role in bringing about vanous types of mutations.
Radioimmunoassays (RIA)
It is an extremely sensitive technique in which antibody or antigen is labelled with a radioactive material. The amount of radioactive material in the antigen-antibody complex can be measured with which concentration of antigen or antibody can be assayed. After the reaction ‘free’ and ‘bound’ fractions of antigen are separated and their radioactivity-measured.
MICROBIAL VIRULENCE FACTORS
Microbial virulence factors are gene products required for a microbial pathogen to establish itself in the host. These gene products are located on the bacterial chromosome, or on mobile genetic elements, such as plasmids or transposons.
Primary pathogens express virulence factors that allow them to cause disease in the normal host.
Opportunistic pathogens are environmental organisms or normal flora that lack the means to overcome normal host defense mechanisms. They cause disease only when the normal host defenses are breached or deficient.
Virulence factors can be divided into several categories.
Skin - Propionibacterium acnes, Staphlococcus epidermis , diptheroids; transient colonization by Staphlococcus
aureus
Oral cavity - Viridans Streptococci, Branhamella species, Prevotella melaninogenicus, Actinomyces species, Peptostreptococcus species, other anaerobes
Nasopharynx Oral organisms; transient colonization by S. pneumoniae, Haemophilus species, N. meningitidis
Stomach Rapidly becomes sterile
Small intestine Scant
Colon - Bacteroides species, Clostridium species, Fusobacterium species, E. coli, Proteus species, Pseudomonas aeruginosa, Enterococcus species, other bacteria and yeasts
Vagina - Childbearing years:Lactobacillus species, yeasts, Streptococcus species
Prepuberty / Postmenopause: colonic and skin flora
A. Enzyme production can be of several types depending on the needs of the organism, its requirements for survival, and the local environment.
1. Hyaluronidase breaks down hyaluronic acid to aid in the digestion of tissue.
2. Protease digests proteins to enhance the spread of infections.
3. Coagulase allows coagulation of fibrinogen to clot plasma.
4. Collagenase breaks down collagen (connective tissues).
B. Toxins
1. Exotoxins are heat-labile proteins with specific enzymatic activities produced by many Gram-positive and Gram-negative organisms. Exotoxins are released extracellularly and are often the sole cause of disease.
a. Some toxins have several domains with discrete biological functions that confer maximal toxicity. An example is A-B exotoxin, where the B subunit binds to host tissue cell glycoproteins and the A subunit enzymatically attacks a susceptible target.
b. Many toxins are ADP-ribosylating toxins
2. Endotoxin is the heat-stable lipopolysaccharide moiety found in the outer membrane of Gram-negative organisms. when released by cell lysls, the lipid A portion of lipopolysaccharide can induce septic shock characterized by fever, acidosis, hypotension, complement consumption, and disseminated intravascular coagulation (DIC).
C. Surface components
may protect the organism from immune responses such as phagocytosis or aid in tissue invasion. For example, the polysaccharide capsules of H. influenzae type b and the acidic polysaccharide capsule of Streptococcus pneumoniae interfere with phagocytosis. Other surface proteins, such as adhesins or filamentous appendages (fimbriae, pili), are involved in adherence of invading microorganisms to cells of the host.
Application of agglutination reactions
Agglutination reaction Example
Tube agglutination -> Widal test, Weil Felix reaction, Standard tube test for brucellosis
Slide agglutination -> Typing of pneumococci,Diagnosis of Salmonella,Diagnosis of Shigella
Agglutination Absorption test -> Salmonella diagnosis
Coagglutination -> Grouping of streptococci, Identification of gonococci, Detection of Haemophilus, Antigen in CSF
Passive agglutination
Latex agglutination Detection of HBs Ag, ASO, CRP